In the first part of the present work, we highlightthe crucial importance of the chemical activation  stepon the performance of Pt–M electrocatalysts in the MEA at highcurrent densities (HCDs). In addition, we provide the scientific communitywith a preliminary and facile method of distinguishing between a “poorly”and “adequately” dealloyed (activated) Pt-alloy electrocatalystusing a much simpler and affordable TF-RDE methodology using the well-knownCO-stripping process. Since the transition-metal cations can alsobe introduced in a PEMFC due to the degradation of the Pt–MNPs, the second part of the work focuses on presenting clear evidenceon the direct impact of the lower voltage limit (LVL) on the stabilityof Pt–M electrocatalysts. The data suggests that in additionto intrinsic improvements in stability, significant improvements inthe PEMFC lifetime can also be obtained via the correctMEA design and applied limits of operation, namely, restricting notjust the upper but equally important also the lower operation voltage.Pt-alloy (Pt–M)nanoparticles (NPs) with less-expensive 3d transitionmetals  supported on high-surface-area carbon supportsare currently the state-of-the-art (SoA) solution to reach the productionphase in proton exchange membrane fuel cells (PEMFCs). However, whilePt–M electrocatalysts show promise in terms of increased activityfor oxygen reduction reaction (ORR) and, thus, cost reductions fromthe significantly lower use of expensive and rare Pt, key challengesin terms of synthesis, activation, and stability remain to unlocktheir true potential. This work systematically tackles them with acombination of electrocatalyst synthesis and characterization methodologiesincluding thin-film rotating disc electrodes (TF-RDEs), an electrochemicalflow cell linked to an inductively coupled plasma mass spectrometer(EFC-ICP-MS), and testing in 50 cm To achieve significantcost reductions, the amount of Pt in the cathode electrocatalyst mustbe reduced. High activity, high electrochemically active surface area(ECSA), and high durability are all necessary for achieving significantimprovements.6 While completely eliminating platinumgroup metals (PGMs) by the use of PGM-free electrocatalysts7 might seem a rational approach to reduce costs,achieving sufficient power densities as well as the durability ofPGM-free electrocatalysts remains a major challenge, with currentlyno clear solution in sight.10 On the other hand, dealloyed Pt-alloy (Pt–M) nanoparticles(NPs) with less-expensive 3d transition metals  supportedon high-surface-area carbon supports are currently the state-of-the-art(SoA) solution to reach the production phase in PEMFCs12—resulting from their facile and already at scale preparation.Cost reduction using Pt-alloys is possible due to two key features:(i) Pt-alloys dilute Pt-atoms inside the nanoparticle’s coreand thus improve Pt overall utilization;16 and (ii) they exhibit a higherkinetic activity toward the ORR due to a combination of a ligand,strain, coordination number, and/or surface disorder effects.23 This class of electrocatalysts has reached significant developmentas part of past Department of Energy (DoE) projects 26 and resulted in record in-MEA activities that exceed the DoE performancetargets27 of >0.44 A/mgPt with<40% activity loss after 30k voltage cycles in the rotating discelectrode (RDE) and MEAs.28 For this classof electrocatalysts, alloying is usually obtained via high-temperature thermal annealing.29Electrification ofthe transport sector and the transition towardrenewable energy solutions are crucial in the race toward cuttinggreenhouse gas emissions. While battery electric vehicles are alreadyleading the way, an important part of the electrification puzzle willbe fuel cell electric vehicles.31 Relatively recently,transition-metal cation contamination in the PEMFC has been recognized as another probablemajor contributor to the poor high current density (HCD) performancewhen using Pt-alloys.35 On the other hand, certain transition-metal cations were shown todiffuse into the membrane, causing formation of hydrogen peroxideradicals (due to the Fenton reaction36)that attack the membrane, leading to failure of the PEMFC. Pt–Cualloys have in the past shown severe durability issues due to protonstarvation of the cathode as a result of Cu plating onto the anodePt/C electrocatalyst and thus blocking the hydrogen oxidation reaction(HOR).38 Similar observations on the presence ofCu in the membrane and in the anode have been reported recently.39 One possible solution to mitigate this has beendemonstrated by Mani and co-workers,40 wherean already fabricated catalyst-coated membrane (CCM) was exposed toanother round of acid washing. However, this solution is not suitablefor any kind of mass production of MEAs. Thus, solutions are necessaryat the level of catalyst production. Consequently, improved fundamentalunderstanding of the influence of chemical activation on the electrochemical behavior of Pt-alloy electrocatalysts is critical.In addition to the importance of the bulk crystal structureofthe Pt-alloy NPs, the dealloying (activation) step of the electrocatalystalso holds crucial importance.41 However,on the topic of metal dissolution, not only the intrinsic propertiesof an electrocatalyst material but also the operational conditionsof the PEMFC seem to play a crucial role. There is important evidencein the literature that highlight the significance of both the uppervoltage limit (UVL) and the lower voltage limit (LVL) in limitingthe degradation of Pt-based carbon-supported electrocatalysts in thePEMFC. For instance, Uchimura and co-workers42 showed that the ECSA loss increases when LVL is reduced from 0.8to 0.6 V when performing accelerated degradation test (ADT) cycleswhile using a fixed UVL (0.95 V). At that time, the transient natureof Pt dissolution was not fully understood and, thus, the mechanisticinterpretation of the increased degradation was not yet consideredsignificant for Pt dissolution arising from the destabilization ofthe Pt-oxide species. This was changed with the introduction of couplingmethodologies between electrochemical cells and an inductively coupledplasma mass spectrometer (ICP-MS) that enabled time-and-potential-resolvedmonitoring of dissolution of metals.46 The investigations revealed asignificant link between Pt dissolution and cathodically reduced Pt-oxideresulting from the so-called oxide-place exchange mechanism.43 Lastly, Yoshida and co-workers47 affirmed already during the launch of the first-generationToyota Mirai the importance of limiting both the UVL and LVL to increasethe fuel cell lifetime. This was later confirmed during the investigationof the first-generation Toyota Mirai stack as part of the Departmentof Energy (DoE) Mirai Fuel Cell Vehicle report, revealing system-levellimitations on the LVL of the stack.48 Whilehigh UVLs occurring during the start-up/shut-down of the PEMFC leadto severe carbon corrosion,49 the LVL seemsto also play a significant role that is perhaps of particular importancefor the successful implementation of Pt-alloys. For this reason, furtherfundamental insights are necessary to truly understand this phenomenon.In addition to the possible introduction of transition-metal cationsin the MEA during the phases of electrocatalyst ink preparation and/orCCM fabrication, the leaching can also occur during the break-in proceduresand/or because of the Pt–M electrocatalyst degradation thatresults in metal dissolution.2 MEAs in single cells under a rangeof conditions. Furthermore, an additional link is established thatconnects the dealloying step with the observed TF-RDE features, in-linemetal dissolution trends coupled to an inductively coupled plasma mass spectrometer (ICP-MS)),and corresponding single-cell performances. In addition, the effectof the LVL on the stability of Pt-alloys toward the dissolution ofthe less noble metal was investigated by a combination of both in-linemetal dissolution measurements and accelerated degradation tests (ADT)in 50 cm2 single-cell data.Thus, in the present work, particular attention was given to theeffect of the dealloying (activation) step on the performance withTF-RDEs and in 50 cm51 Briefly, the electrocatalysts were prepared in three steps. In thefirst step, Pt NPs were deposited onto a commercial carbon black support(Ketjen Black EC300J) via the double passivationgalvanic displacement method reported elsewhere.50 In the second step, the prepared composites with depositedPt NPs were thermally annealed to obtain an alloy crystal phase. Inthe last step, the electrocatalysts were subjected to two differentactivation (acid washing) protocols, thus forming a Pt-rich overlayer via dealloying. Within this study, the Pt-rich overlayershould be understood that the topmost layers of the Pt-alloy NPs havea more Pt-rich chemical composition than the bulk of the Pt-alloyNPs. In other words, the Pt-rich overlayer does not indicate an ideal“core–shell” structure52 and still contains some of the less noble metal.41 The first protocol was performed in accordance with a workdescribed previously.30 Briefly, the processinvolves four times washing in 1 M acetic acid under CO purge. Thesetwo electrocatalysts are denoted as d-Pt-Cu/C-NIC-A and d-Pt-Ni/C-NIC-A,and the activation protocol is denoted as A-activation. The secondprotocol involves 24 h washing in 0.5 M H2SO4 at 80 °C. This protocol was developed during the previous DoEprojects.26 These two electrocatalysts are denoted as d-Pt-Cu/C-NIC-Sand d-Pt-Ni/C-NIC-S, and the activation protocol is denoted as S-activation.The results within this study are then compared to the activated d-Pt-Ni/C-JMbenchmark with a similar metal loading provided by Johnson Matthey(JM).The Pt–M (M = Ni or Cu) electrocatalystswere prepared in accordance with the processes already reported previously.Thepowder X-ray diffraction (XRD) measurements of all samples were carriedout on a PANalytical X’Pert PRO MPD diffractometer with CuKα1 radiation (λ = 1.5406 Å) in the 2θ rangefrom 10 to 60° with the 0.034° step per 100 s using a full-openedX’Celerator detector. Samples were prepared on a zero-backgroundSi holder.Scanning transmission electron microscopy (STEM) imagingwas carried out in a probe Cs-corrected scanning transmission electronmicroscope Jeol ARM 200 CF operated at 80 kV.The samples were examined in the JEM 2800 (scanning)transmission electron microscope using the following instrumentalcondition: voltage (kV) 200.–1, Milli-Q, Millipore) and ultrapure acids  were used. Standards were prepared in-house by dilutionof certified, traceable, inductively coupled plasma (ICP)-grade single-elementstandards (Merck CertiPUR). A Varian 715-ES ICP optical emission spectrometerwas used. Prior to ICP-OES analysis, each electrocatalyst was weighted(approximately 10 mg) and digested using a microwave-assisted digestionsystem  in a solution of 6 mL of HCl and 2 mLof HNO3. Samples were then filtered, and the filter paperwas again submitted to the same digestion protocol. These two timesdigested samples were cooled to RT and then diluted with 2% v/v HNO3 until the concentration was within the desired concentrationrange.All reagents used wereof analytical grade or better. For sample dilution and preparationof standards, ultrapure water  electrolyte with a conventional three-electrodesystem. Ag|AgCl was used as a reference, and a graphite rod was usedas a counter electrode. The working electrode was a glassy carbondisc embedded in Teflon (Pine Instruments) with a geometric surfacearea of 0.196 cm2. The Ag|AgCl reference was separatedfrom both the working and counter electrodes with a salt bridge toavoid Cl–-ion contamination. Prior to each experiment,the two-compartment electrochemical cell was boiled in Milli-Q waterfor 1 h, and the electrode was polished to mirror finish with Al2O3 paste on a polishing cloth (Buehler). After polishing, the electrodes wererinsed and ultrasonicated (Ultrasound bath Iskra Sonis 4) in a Milli-Q/isopropanolmixture for 5 min. Then, 20 μL of 1 mg mL–1 water-based well-dispersed electrocatalyst ink was pipetted on theglassy carbon electrode completely covering it and dried under ambientconditions. After the drop had dried, 5 μL of Nafion solution diluted in isopropanol (1:50) wasadded. The electrode was then mounted on the rotator (Pine Instruments).The Ag|AgCl reference electrode potential against the reversible hydrogenelectrode (RHE) was determined before the start of the experiment.Electrochemical measurements were conductedwith a CompactStat(Ivium Technologies) in a two-compartment electrochemical cell ina 0.1 M HClORHE. The electrocatalyst was electrochemicallyactivated ) for 200 cycles between0.05 and 1.2 VRHE with a scan rate of 300 mV s–1 at 600 rpm. After PCA, the electrolyte was exchanged with a freshone and the electrode was no longer under any external potential control. ORR polarization curves were measured in anoxygen saturated electrolyte at 1600 rpm in the potential window 0.05–1.0VRHE with a scan rate of 20 mV s–1. Ohmicresistance of the electrolyte was determined and compensated for asreported in ref (RHE) to ensure successful CO adsorption. Afterward, the electrolytewas saturated with Ar. CO-electrooxidation was performed using thesame potential window and scan rate as in ORR, but without rotationand in an Ar saturated electrolyte. After subtraction of the backgroundcurrent due to capacitive currents, kinetic parameters were calculatedat 0.9 VRHE using the Koutecky–Levich equation.54 Electrochemically active surface area (ECSACO) was determined by integrating the charge in CO-electrooxidation(“stripping”) experiments as described in ref  electrocatalyst measurements, the electrodewas placed in an Ar saturated electrolyte under potential controlat 0.05 Vd in ref . At the d in ref . All potRHE, 300 mV s–1, 600 rpm) were performedunder the same parameters as in the case of the Pt/C benchmark electrocatalyst.Likewise, the electrolyte was exchanged with a fresh one at the end.ORR polarization curves and CO-electrooxidation were measured onceagain under the same conditions as before PCA. Each Pt-alloy electrocatalystwas measured at least three times.The electrocatalystswere placed in the electrolyte without any potential control (at theOCP) in an oxygen saturated solution, and ORR polarization curveswere measured immediately after measurement of ohmic resistance andits compensation . The protocol for CO-electrooxidationfollowing the ORR measurement was the same as described above. Atthe end of CO-electrooxidation, 50 cycles of PCA  were glassy carbon discs (3 mm diameter) embeddedinto a polyether ether ketone (PEEK) material (BASi). The discs werealigned in series; the counter electrode was placed first and theworking electrode second in the direction of the electrolyte flow.The sample was deposited on the electrode by drop-casting a 5 μLdrop of the ultrasonically homogenized catalyst ink (1 mg mL–1). Such a preparation resulted in an electrocatalyst loading of 5μg for all electrocatalysts. In addition, to increase the surfacearea of the counter electrode, a 5 μL drop of the Ketjen BlackEC300J suspension (1 mg mL–1) was deposited on theglassy carbon counter electrode. After the drop had dried, 5 μLof Nafion solution  diluted in isopropanol(1:50) was added. The Ag|AgCl reference electrode potential againstRHE was determined before the start of the experiment. The housingof the cell was made from PEEK material, and the design was modeledafter a commercial cross-flow cell .The volume of the cell was established with a home-made silicon gasketwith 1.0 mm thickness and a 1.5 cm2 ellipsoidal cut. Thecarrier solution (0.1 M HClO4) was pumped through the cellat a constant flow of 400 μL min–1. Two glasssyringes using Luer Lock connection to a poly(tetrafluoroethylene)(PTFE) tubing, two syringe pumps (WPI AL1000-220Z), and a diagonalfour-way flow valve  were used to enable a continuousflow of the solution.The setup and measurement guidelines were establishedas part ofprevious works 1.30,31,4–1; makeup 0.28 L min–1; plasma 1 L min–1; and cooling15 L min–1. The signals were recorded for Cu63, Ni60, and Pt195 with 0.5 s integrationper data point. To convert the ICP-MS signals to concentration (ppb),standard solutions of Cu, Ni, and Pt in 0.1 M HClO4 wererecorded with the following concentrations: 0.5, 1, 2, 5, 10, 20,50, and 100 ppb.The EFC was coupled withan ICP-MS detector, namely, Agilent 7900ce ICP-MS instrument , equipped with a MicroMist glass concentricnebulizer and a Peltier cooled Scott-type double-pass quartz spraychamber. A forward radio-frequency power of 1500 W was used with Argas flows: carrier 0.85 L minFigure S1, Supporting Information (SI). Initially,Milli-Q water was pumped through the cell under open-circuit conditions(OCPs) before switching to 0.1 M HClO4. After 10 min ofacid flow, the potentiodynamic protocol was started; to check forthe effect of the lower potential limit (LPL), the electrocatalystswere cycled for three cycles between 0.925 and X VRHE  with nine cyclesin total (scan rate of 5 mV s–1). In another setof experiments, “memory effect” was checked by omitting X = 0.7 and 0.65 and only performing three cycles between0.925 and 0.6 VRHE. In both cases, the experiment was followedwith two cycles between 0.05 and 1.4 VRHE (scan rate of5 mV s–1). After each experiment, a sequence ofpotential pulses  wasperformed to synchronize the electrochemical experiment with the ICP-MSsignal. Briefly, at the end of the experiment, the system is leftat OCP for 200 s for the MS signal to reach background levels. Thisis followed by applying the first potential (oxidative) “pulse”(for 0.5 s) until 1.4 VRHE. After additional 150 s of waitingat OCP, a second potential (reductive) pulse (for 0.5 s) of 0.05 VRHE is applied. Both pulses result in significant dissolutionin the ICP-MS data and enabling one to observe the time differencebetween the electrochemical pulse and the detection of the metal dissolutionon the MS.Electrochemicalexperiments were performed with a CompactStat (Ivium Technologies)with a typical three-electrode setup. No ohmic drop compensation methodwas used. The general electrochemical protocol is presented in ca. 0.9 and metal loadings of ca. 0.10 mgPt cm–2, unless specified otherwise.The anode catalyst layer was kept constant at an ionomer/carbon weightratio of ca. 1.5/1 and a metal loading of 0.1 mgPt cm–2. The membrane used was a perfluorosulfonicacid type, fabricated at JMFC with a thickness of ca. 20 μm. Catalyst layers were produced on a PTFE substrate andtransferred via a decal method onto the membrane.Single cells (50 cm2 active area) were assembled by sandwichingthe catalyst-coated membranes between the gas diffusion layers (GDLs)and applying an average compression onto the active area.The MEAs used inthis work consist of five layers. Nafion 1100EW  was used to fabricate thin-layer electrodes.The cathode catalyst layers were prepared at an ionomer/carbon weightratio of 2 as the anodereactant . Stoichiometric flow rates ofanode (s = 2) and cathode (s = 9.5for O2 and s = 2 for air) reactants wereused at current densities >0.2 A cm–2 and constantflows (corresponding to 0.2 A cm–2 flows) at <0.2A cm–2. Reactant humidification was achieved bywater-bubblers, the temperatures of which were calibrated to yieldthe desired relative humidity (RH) values. Humidity and cell pressurewere measured at the inlet for both electrodes. Cell resistances asa function of current density  were determined usingHioki at 1 kHz. Multiple-path serpentine flow-fields  machinedinto sealed graphite blocks were used for testing.The fuel cellstation was built in-house at JMFC. Pure oxygen and synthetic airwere used as cathode reactants and pure H–2 under H2/Air at 50 kPagauge, 100% RH, and 80 °C. The cell voltage was monitored untila stable value was observed. The conditioning step lasted 2 h unlessspecified otherwise. Afterward, the cathode catalyst layer was exposedto a series of cathode starvation steps (see below) followed by a2 h current hold at 500 mA cm–2 until a stable voltagewas observed. After the starvation steps, the MEA was ready for testingby a series of H2/O2 polarization curves formass activity (MA) quantification at different stages of the protocol. The polarization curves wererecorded from low  to high current  ascending direction and backward, descending direction. The currentdensity was maintained for 3 min at each step, and the MA value wasobtained from the ascending polarization curve at 0.9 V by extrapolationresistance correction. H2-crossover current densities weremeasured using the procedure described by Kocha et al.60 In this test, the hydrogen that permeates throughthe membrane to the cathode is oxidized by the application of a voltage, and the resulting currentwas measured. Therefore, the cell was operated under H2/N2, and the gas crossover measurements were done at eachof the operating conditions . The catalyst activities were evaluatedbased on H2-crossover corrected current densities, ieff . MA values reported in this article were not corrected for H2-crossover because the measured crossover currents accountedfor a maximum of 10% at the loadings used in this study.The MEAswere conditioned by the application of a constant current densityof 500 mA cm2 at 300 mL min–1 for 15 min. Afterward, the cathodewas purged with N2 at the same flow rate for 2 h to ensurethat CO is removed from the bubblers and the catalyst layer pores.The adsorbed CO is oxidized electrochemically by scanning the cathodevoltage from 0.125 to 0.85 V and back to 0.05 V, at 20 mV s–1 for three cycles. The area under the CO oxidation peak is integratedby subtracting the third scan from the first scan and using a 420μC cm–2 constant for a CO monolayer on Pt.TheECSA was measured with the CO-stripping method using the cellin the half-cell mode where the anode electrode acts as a pseudo-referenceelectrode. The cathode voltage was controlled at 0.125 V at 80 °C,100% RH, and 50 kPa gauge while purging with 1% CO in N2/N2 with the upper potential limit (UPL) fixed at 0.925 V, whilethe LVL was changed to 0.5, 0.6, or 0.7 V. At both LVL and UVL, thevoltage was held for 3 s, while the rise time in between the voltagewindow of (LVL–0.925) V was below 0.5 s. The durability protocolwas applied for a total of 1000 cycles, at 80 °C, 100% RH, andambient pressure at the cell outlet. Afterward, the cell voltage wasmeasured at 1.2 A cm–2 at different temperaturesas described in the text.For the voltage window experiments, the benchmark d-Pt-Ni/C-JMcathode catalyst was tested for durability under H51 In the second step (29 In the last step (g30) that uses mild conditions such asacetic acid, but in combination with an adsorptive gas such as COg. The adsorptive gas binds to the Pt-surface and inhibitsany possible readsorption of redeposition of M ions  onto the Pt-surface. This improvesthe ability to wash the dealloyed M out of the carbon matrix duringthe process of acid washing.30 Hereinafter,we will refer to this acid-washing activation as protocol “A”and the electrocatalysts d-Pt-Ni/C-NIC-A and d-Pt-Cu/C-NIC-A. On theother hand, the second dealloying activation protocol included theuse of a stronger acid such as sulfuric acid (H2SO4), also reported by others as part of previous DoE projects.26 Hereinafter, we will refer to this acid-washing activation as protocol“S” and the electrocatalysts d-Pt-Ni/C-NIC-S and d-Pt-Cu/C-NIC-S.Furthermore, the results within this study are compared to the d-Pt-Ni/C-JMbenchmark with a similar metal loading provided by Johnson Matthey(JM).ond step 1b, the oast step 1c, for tFigures S3–S5, SI) provide evidence ofa very high loading and uniform density of NPs over the carbon support.Additionally, a closer inspection reveals the detailed structure ofseveral NPs, which include a Pt-rich overlayer . In addition, the observed width of the main X-ray diffraction(XRD) peaks  phase is also observed with both TEM and XRD. In the caseof the “A-activated” Pt–Cu catalyst  characteristicof the 1:1 (Pt–Cu) ratio is observed.61 On the other hand, the A-activated Pt–Ni catalyst  peaks 1d,e corrcatalyst 1d, a smacatalyst 1e exhibicatalyst 1e. Firsttivation1e also rtivation1d, the ptivation1e.CO) peak, while the kinetic performance for ORR was measuredat both 0.9 and 0.95 VRHE .55 The liquid half-cell TF-RDEperformance was evaluated to confirm the eligibility of the Pt–MNIC electrocatalysts for further investigation in 50 cm2 single cells. This required the catalysts to exhibit a superiorkinetic ORR performance in contrast to SoA Pt/C electrocatalysts64 as well as an ECSACO higher thanat least 40 m2 gPt–1.5 According to expectations, the liquid half-cellTF-RDE kinetic performance of all Pt–M NIC catalysts at both0.9 and 0.95 VRHE exceeds that of Pt/C from Tanaka KikinzokuKogyo . In addition,the measured ORR performances are comparable to or even exceed thatof the d-Pt-Ni/C-JM benchmark. Lastly, evaluation of ECSACO for all Pt–M NIC electrocatalysts also confirmed the suitabilityfor further evaluation in single-cell measurements at JM testing facilities.Figures S8–S10, SI). In the case of TF-RDEmeasurements, both A- and S-activated catalysts were first evaluatedby measuring the ORR  followed by CO-electrooxidation . This was then followed by an additional 50 electrochemical cyclesof potential cycling activation . After PCA,ORR and CO-electrooxidation were measured under the same conditionsonce again.Prior to the single-cell evaluation,a more in-depth analysis wasconducted to provide a baseline understanding of the differences betweenA- and S-activation protocols on both the liquid half-cell TF-RDEelectrochemical properties as well as in-line metal dissolution , the peak maximum corresponding to CO-electrooxidationat approximately 0.8 VRHE shifts toward a more positivepotential for ∼24 mV. However, for the case of more harshlyactivated d-Pt-Ni/C-NIC-S . In addition, when comparing the 2nd (start)and 50th (end) cyclovoltammograms (CVs) of the PCA, a slight changein the distinct features corresponding to the Pt-surface such as thehydrogen underpotential deposition (HUPD) region and Pt-oxideformation and reduction regions can be observed . When comparing the changes in these featuresfor both A- and S-activated catalysts, respectively, unlike with thedifference in the CO-electrooxidation maximum shift, no major differencedistinguishing both activation protocols is revealed. Interestingly,the exact same 10 mV difference in the CO-electrooxidation peak maximumbefore and after PCA was observed in the case of the d-Pt-Ni/C-JMbenchmark . However,similar to both A- and S-activated Pt–Ni catalysts, PCA againhad no effect on the ORR polarization curves corresponding to theirkinetic performances measured in half-cell TF-RDE before and afterPCA . In addition,the changes in Pt-surface features when comparing the 2nd (start)and 50th (end) CVs of the PCA are also similar ./C-NIC-S 2b, this Figure S10a,b, SI). Lastly, also the comparisonof distinct features corresponding to the Pt-surface in the 2nd and50th CVs of the PCA  reveals only slight changes for both A- and S-activated Pt–Cucatalysts, respectively. The take-away message here is that followingour TF-RDE protocol, only a slight difference in the shift of theCO-electrooxidation peak maximum provides a possible clue that theA- and S-activated catalysts could in fact be quite different despiteexhibiting a similar ORR activity in TF-RDE.To confirm that the observed effectsare not a feature of the Pt–Nialloy system, the exact same TF-RDE protocol was also performed onthe d-Pt-Cu/C-NIC-A and d-Pt-Cu/C-NIC-S electrocatalysts 2c,d. Int65 highly sensitive method, namely, electrochemical flow cell (EFC)coupled to an inductively coupled plasma mass spectrometer (ICP-MS).This advanced electrochemical characterization method allows us totrack on-line time-and-potential-resolved dissolution of metals . Other variations of the methodologyused by other research groups, instead of the EFC, include usage ofa scanning flow cell (SFC)67 or a variation used in combination with an RDE system.69 The intrinsic metal dissolution was monitored in accordance withthe protocol presented in Figure S1. Briefly,each Pt-alloy electrocatalyst was exposed to the open-circuit potentialconditions (I. OCP). This was followed by evaluation of the lowerpotential limit (II. LPL) effect  that will be presented indetail later on. Lastly, the LPL protocol was followed by two morewide potential window (III. WPW) cycles between 0.05 VRHE and the upper potential limit (UPL) of 1.4 VRHE  to initiate substantial dissolution ofPt and, consequently, also dissolution of the less noble metal. Inother words, we wanted to probe how much of the less noble metal getsdissolved if we severely damage the “stable” Pt-richsurface. If parts II. and III. would be reversed, the WPW cycles (0.05–1.4VRHE) could already substantially deplete the intrinsicallyunstable less noble metal from the Pt-rich overlayer and highly affectthe results of the LPL studies presented in the next chapter. Thisis because the quantities of metal dissolution are substantially higherwhen Pt-based electrocatalysts are exposed to a wider potential window.67We can get thefirst confirmation of these differences by measuringthe respective intrinsic metal dissolution profiles of activated Pt-alloyelectrocatalysts using a well-establishedRHE, 5 mV s–1, 0.1 M HClO4; Figure S9d, SI), one can expect two typical transientPt dissolution peaks.72 Usually, this includes a smaller peak correspondingto the anodic (A1) and a more dominant cathodic (C1) Pt dissolution.The anodic (A1) Pt dissolution mechanism involves surface structureroughening caused by the oxide-place exchange mechanism.43 This creates Pt defects (low coordination sites),which do not get passivated by oxide formation and are thus proneto dissolution.45 On the other hand, withfurther penetration of oxygen in the crystal lattice of the NPs, reductionof this Pt-oxide results in formation of a much larger amount of unstablePt defects, leading to a significant cathodic (C1) dissolution.43 Furthermore, what seems to be a general featurefor all Pt-alloys41 is that following boththe A1 and C1 Pt dissolution, we also observe A1′ and C1′less noble metal dissolution. In other words, every time Pt dissolves,this exposes previously protected M atoms and causes their subsequentdissolution.31 In addition to that, Pt-alloysusually also exhibit another peak related to the less noble metaldissolution (A2′). However, what is special in this case isthat A2′ does not have a corresponding Pt dissolution peaksuch as A1′ and C1′. For instance, the A2′ Cudissolution peak .41 On the other hand, the origin of the A2′ Nidissolution peak  cannot be yet determined accurately. What we can presume,however, is that in both cases, the alloyed less noble metal is stableuntil Pt-oxide  is reduced.73 In accordance with the protocol presented in Figure S1 of the SI, after the end of II. LPL,the electrocatalyst is briefly exposed to OCP conditions, followedby a jump to 0.05 VRHE (where the Pt-oxide gets reduced)and the start of WPW cycles. While A2′ Cu  starts todissolve already at the beginning of the cycle (0.05 VRHE). Thus, we can claim that the A2′ dissolution definitelyoccurs on the Pt-alloy NPs. A last but also important difference tonote in relation to the WPW cycles  is comparing the differences in A1′and C1′ dissolution intensities. While both d-Pt-Ni/C-NIC-S exhibit a higher intensity of C1′ Ni dissolution than A1′,the situation is reversed in the case of d-Pt-Cu/C-NIC-S . A slight difference most likely originates due to the slightlyhigher metal (Pt and Ni wt %) loadings in the case of d-Pt-Ni/C-NIC-S. Analogously, also comparisonof d-Pt-Cu/C-NIC-A , we argue that the potentialdifference in the CO-electroxidation peak maximum can serve as a verysimple and sensitive preliminary indicator that can help distinguishbetween a poorly or adequately dealloyed (activated) Pt-alloy electrocatalyst.The effect of the less noble metal ions on the position of the CO-electrooxidationpeak has already been explored on the Pt/C electrocatalyst by Durstand co-workers.75 In their study, theyspiked the electrolyte with different amounts of various metal ionsand observed shifts in the position of the CO-electrooxidation peaktoward more negative potentials. This occurs because less noble metalions induce a more facile (hydr)oxide formation at the Pt-surfacein the double layer. Hydrated less noble metal cations are locatedbetween the inner and outer Helmholtz planes where they partiallylose their hydration shell and come closer to the Pt-surface, inducingan increase in the OHad coverage that can cause the shiftin the onset of CO-electrooxidation toward lower potentials.75 Following this study, we explored this on Pt-alloysas part of our previous work where also a more “mild”electrochemical activation  resulted ina shift of the CO-electrooxidation peak maximum toward lower potentialsin comparison to a harsher one .31 However, the present study, for the first time, provides the necessarydata that correlates this effect with “real” electrocatalystsamples based on the used chemical activation protocol (A- or S-activation).Thus, if our assumptions are correct, a similar correlation shouldbe observed when performances of A- and S-activated Pt–M NICanalogues are evaluated in the 50 cm2 single cells.Because single-cell testing is time consuming, requiresrelativelylarge amounts of the electrocatalyst (in contrast to TF-RDE), andis highly complex in nature (many parameters can influence performance),it is highly important to be able to have a preliminary and facilemethod of distinguishing between a “poorly” or “adequately”dealloyed (activated) Pt-alloy electrocatalyst. Providing such a solutionhas been one of the core goals of this study. Namely, the data sofar suggests that both A-activated analogues not only exhibited alarger shift in the potential of the CO-electrooxidation peak maximum2a–dbut CO (at 100% RH) for all investigated electrocatalystsin a 50 cm2 single cell (MEA) is presented in 38). On the otherhand, the kinetic performance of d-Pt-Ni/C-NIC-A is also worse thanthat of the S-activated catalyst despite the trend being reversedin TF-RDE  andECSAn TF-RDE 1.In addprotocol 1e. This protocol 1.In conprotocol 1, where protocol 2 is alre2 single-cell ORR polarization curves and, thus, HCDperformances at various conditions  and “hot-dry” conditions. 2 single-cell ORR polarization curves between d-Pt-Ni/C-NIC-Aand d-Pt-Ni/C-NIC-S electrocatalysts in hot-wet conditions in bothO2 and air. Interestingly, no significant difference isobserved in the O2 performance for both A- and S-activatedcatalysts up until 2 A cmgeo–2, whereasimpressively, the voltage did not yet drop below 0.7 V. In contrast,in accordance with expectations, performance of both A- and S-activatedcatalysts drops significantly when O2 on the cathode isexchanged for air. Nevertheless, both catalysts still achieve an impressive1.4 A cmgeo–2 at 0.6 V with the cathodeloading of the S-activated catalyst being only ∼82 μgPt cm–2  “Tolerance” for Niions in single cells far exceeds that of Cu ions. When comparing bothA-activated Pt-alloy catalysts at hot-wet conditions .This mainly suggests three things.(i) Comparing Pt-alloy cathodesat both wet and dry conditions can reveal performance differencesrelated to the presence of the less noble metal ions in the MEA. Inparticular, the present data also suggests that measuring only atwet conditions could even be misleading and mask transport issuesrelated to the presence of less noble metal ions. Thus, measuringat a lower (dryer) RH as well as in air rather than in Onditions 3a,c, d-Pnditions 1e that mnditions 2, S-acti72 as well as on the dissolution of the less noble metalin the case of Pt-alloys65 has already been well documented. However, most of the existingstudies focus on the understanding of the changes in UPL of 1.0 Vor above . On the other hand, understanding the changesin LVL,42 especially at UVLs below 1.0V, is perhaps similar to the electrocatalyst activation, vastly underestimatedand understudied.72 Considering that the fuel cellstack will operate for the vast majority of its time within a voltagewindow of around 0.6–0.95 V, understanding the mechanism ofPt and Pt-alloy degradation in this regime is in fact critical.The effect of the voltage window on Pt dissolutionX VRHE; X = 70/65/60, 5 mV s–1, 0.1 M HClO4) and evaluating all fivePt-alloy electrocatalysts from this study. The experimental protocolconsists of in total nine slow cycles with a scan rate of 5 mV s–1 in the operational potential window with a constantupper potential limit (UPL) and a decreasing LPL for 50 mV every threecycles. RHE on the dissolution of both Pt and Ni inthe case of the d-Pt-Ni/C-JM benchmark. Interestingly, while we havekept the UPL constant, lowering of the LPL results in an increaseof Ni dissolution. Furthermore, we have tested this effect for reproducibility as well as excludedthe possibility of a memory effect. Thus, in addition to the reproducibilitymeasurements, where the initial LPL is 0.7 VRHE , we performed two more measurementswhere the LPL of 0.6 VRHE was used right away . This way, no prior metaltransient dissolution and, thus, degradation took place prior to thethree cycles with an LPL of 0.6 VRHE. By going directlyto the lowest LPL of 0.6 V , the observed Ni dissolution was indeed substantially higherthan in the case with an LPL of 0.7 VRHE .Thus, we are hereby providing valuable evidence on the effect ofthe lower potential limit (LPL) on metal dissolution by initiallyusing the EFC-ICP-MS . The results clearly indicatethat the highest voltage losses were observed in the case of ADT conditionswith an LVL of 0.50 VRHE, whereas the voltage losses decreasedramatically if the LVL is increased to 0.60 or 0.70 VRHE. The trends presented in 71 Consequently, at the same LVL, a higher fractionof Pt-oxide gets reduced with increasing temperature, leading to alower protection of the less noble metal toward the dissolution and,thus, a higher voltage loss. The effects of both the voltage windowand the temperature are in line with our recent liquid electrolytefollow-up study.74To provide further evidence onthis important phenomenon, we extendedour study by performing carefully designed accelerated degradationtests (ADTs) in 50 cm48 with a detailed investigation of the operatedMirai stack showed that under system-controlled operation the voltagewindow was kept within the range 0.65–0.85 V. Analysis of MEAsshowed no Co dissolution into the membrane under system-controlledoperation, but that a large amount of Co had dissolved after the applicationof the ADT for catalyst durability defined by the US-DoE 0.60–0.95V voltage window. The reported cathode loading for the Mirai MEAsunder study was ca. 0.30 mgPt cm–2, and under the operating conditions used, this loading was enoughto prevent exposure to aggressive upper and lower voltages . It is plausible that thisbenignvoltage window leads to low Pt dissolution and is probably one ofthe causes that prevented significant dissolution of cobalt. Clearly,this provides solid evidence that the voltage window used to determinethe durability of Pt-alloys is extremely important.As further evidenceon the importance of the results in the presentstudy, revealing data published by the Argonne National LaboratoryFigure S1 for the II. LPLpart of the EFC-ICP-MS protocol). First and foremost, we observe theexact same effect  as in the case of the d-Pt-Ni/C-JM benchmark(S8–S10), the only noticeable difference between the A- and S-activatedcatalysts for both Pt–Ni and Pt–Cu catalysts was observedin the shift of the CO-electrooxidation peak maximum measured beforeand after PCA. However, following also the additional comparison ofthe metal dissolution trends  used in this work, can be of particular importance to enablealloy cathodes. Of course, the voltage at which full Pt-oxide reductionoccurs depends on the T, RH, and p used in addition to the composition of the cathode catalyst layer.The observations derived from this work lean toward the hypothesisthat Pt-alloys are particularly prone to faster degradation comparedto pure Pt if exposed to voltage windows where almost all of the Pt-oxideand OHad are removed and grown again using a dynamic profile,leading to the depletion of the less noble metal in the first threeor four atomic layers and the presence of leached metal ions in theionomer. The explanation is based on the hypothesis that oxygen diffusioninto the subsurface is an important step in the formation of the surfaceoxide and for the subsequent Pt dissolution. For a detailed discussionon this topic, the reader is directed to the elegant work by Balbuenaand co-workers77 on the mechanism of subsurfaceoxygen formation, surface segregation, and Pt dissolution for Pt andPt-alloys. Considering that the less noble metal composition in thesecond and third atomic layers of a Pt-alloy is usually in the rangeof Pt/X (3:1) X = Ni, Co, or Cu, from the voltage windows providedin this work, one could easily explain the fast depletion of the lessnoble metal in the subsurface if exposed to the 0.6–0.925 Vvoltage window. Dissolution of the less noble metal is influencedby the reactions that occur in the vicinity of the Pt-oxide growthand reduction. On the other hand, if the catalyst surface is leftslightly oxidized, such as with the use of narrower voltage windows0.70–0.925 V, then the depletion of the less noble metal inthe subsurface is inhibited under the protocol and conditions usedin this work. One would also have to acknowledge that the onset ofthe oxide growth is shifted to higher voltages/potentials for thecase of Pt-alloys compared to Pt.78As discussed in this paper,if the upper voltage is controlledat 0.925 V or lower, the LVL where almost full reduction of the Pt-oxideoccurs,79This is of particular relevance for industrial application in MEAsbecause by preventing the segregation of the less noble metal thenegative impact of dissolved metal ions is avoided. This is oftenoverlooked in wet electrochemical cells where dissolved metal ionsin the electrolyte do not lead to a decrease in ORR activity. However,in MEAs, it is well known that metal ions can diffuse from thecathode layer to the membrane and back again due to the flux of ionsback and forth from the cathode layer to the membrane, proportionalto the flux of protons and hence the load. As a consequence, protonand oxygen transport can be severely decreased in the presence ofdissolved metal ions, which are two of the main reasons for the decreasein performance at high current densities after cycling Pt-alloys,among other factors, as reported by Ramaswamy and co-workers.2 single cells at both the hot-wet  and the hot-dry  conditions. Inthe present study, both A-activated analogues performed significantlyworse in 50 cm2 MEAs, experienced a higher amount of lessnoble metal dissolution, and most importantly, exhibited a largershift in the potential of the CO-electrooxidation peak maximum. Thus,a much simpler and more accessible technique like TF-RDE as a ratherprecise and very sensitive preliminary indicator (via CO-electrooxidation) can help distinguish between a poorly or adequatelydealloyed (activated) Pt-alloy electrocatalyst. On the other hand,also following the protocols used in this study, one can gain insightsinto the chemical activation in relation to the less noble metal dissolutionusing the EFC-ICP-MS. Furthermore, evaluating Pt-alloy electrocatalystsin single cells at a lower RH (hot-dry) as well as in air rather thanin O2 reveals significantly more information about theperformance of Pt-alloy electrocatalyst performance than at high RH(hot-wet). Overall, it is clearly presented that the presence of theless noble metal ions in the CCM is one of the most critical parametersgoverning the performance of Pt-alloys.In summary, to showthe critical importance of the catalyst activation,we have prepared four dealloyed  Pt–M(M = Cu or Ni) electrocatalysts and compared them with the Pt–Nialloy catalyst from Johnson Matthey. Namely, both Pt-alloys were subjectedto either a “milder” acetic acid activation protocolor a “harsher” sulfuric acid activation protocol. BothA- and S-activated analogues were then evaluated using TF-RDE, in-linemetal dissolution using the EFC-ICP-MS methodology, as well as testedin the 50 cm2 single cells provides clearevidence on the significant importance of the LVL/LPL in relationto the less noble metal dissolution. Thus, better understanding ofPt-oxide formation and reduction in relation to Pt dissolution aswell as subsequent dissolution of the less noble metal is crucialto enable the use of Pt-alloy cathodes at HCDs needed for automotiveconditions. We show that operating Pt-alloys below 0.7 V results ina significantly higher degree of less noble metal dissolution and,thus, higher voltage losses when using Pt-alloy cathodes in singlecells even at relatively low UVLs/UPLs of 0.925 V. In addition, wediscovered that oxide-place exchange responsible for Pt cathodic transientdissolution plays a crucial role in this. Being previously consideredas highly important only at UPLs of above 1.1 V, our evidence suggeststhat it is in fact the dominant mechanism responsible for the observedperformance losses already at operating voltages relevant for operationof Pt-alloys in PEMFCs. Since transient dissolution of the less noblemetal is highly connected with the transient dissolution of Pt, followingthis logic, perhaps operational strategies at the system level47 as well as modifications inhibiting Pt dissolution and improvingthe protection of the less noble metal should be of a much largerfocus in the future . Thus, in our opinion, there are still large opportunities notonly in the investigation of novel chemical activation protocols butalso in the development of highly stable high metal loaded/high ECSAPt-alloy electrocatalysts and also in how system-level operation ofPt-alloy electrocatalysts should be viewed in contrast to traditionalPt/C for extending the long-term performance benefits of this stillintriguing class of PEMFC electrocatalysts.In the second part ofthe study, the data generated using bothEFC-ICP-MS and ADTs in 50 cm


Candida albicans of a denture-relining material coated with zinc-bound phosphate-based glass. First, zinc-bound phosphate-based glass was fabricated. A polymerized denture-relining disk was coated with zinc-bound phosphate-based glass . The surface properties of the control and experimental groups were measured, including the wettability, microhardness, color difference, and gloss. The biocompatibility was evaluated using the MTT assay according to ISO 10993-5. The antifungal activity was investigated by counting the number of colony-forming units of Candida albicans. The results were analyzed using a one-way ANOVA and Tukey’s test (p = 0.05). The results of this study indicate that, despite the antimicrobial effect of zinc-bound phosphate-based glass, a coated denture-relining material does not degrade the surface properties and biocompatibility. Therefore, this novel material is considered promising for use as a dental material with antimicrobial properties that can potentially prevent denture stomatitis.This study investigated the surface properties, biocompatibility, and antifungal activity against  Improvements in medical technology and living standards as well as a greater focus on health have led to a steady increase in human lifespan . ConsequCandida albicans [Candida includes the disinfection of dentures, the application of antifungal agents, and medication [Many older adults use dentures ,4. Acrylalbicans . It is kalbicans . The tredication . Howeverdication . In addidication . Accordidication ,17. In adication .Recently, research on natural inorganic antimicrobial substances has been conducted to overcome the resistance caused by antimicrobial abuse ,20, inclAlthough studies on the antimicrobial properties of zinc-doped composites have been conducted in several academic fields ,25, stud2O5 (42 mol%), CaO (25.2 mol%), Na2O (16.8 mol%), and ZnO (16 mol%) powders in a tubular shaker-mixer. The blended batch was melted in an alumina crucible at 1100 °C for 1 h in an electric furnace. The melted glass was then quenched at room temperature to obtain a glass cullet, ground in an alumina mortar, and pulverized under dry conditions using a planetary mono mill .Batches of zinc-doped phosphate glass powder were fabricated by mixing specific weights of high-purity PZn-PBG powder was incorporated in a light-curable coating resin material  and mixed using a magnetic stirrer for 48 h. The following control and experimental groups were prepared: 0 wt% (control), 2 wt% (Zn-PBG-2%), 4 wt% (Zn-PBG-4%), and 6 wt% (Zn-PBG-6%). A Teflon mold (10 mm × 10 mm × 0.1 mm) was positioned on a glass slide covered with transparent polyethylene. The powder and liquid components of the autopolymerizing denture-relining material  were mixed according to the manufacturer’s instructions. The powder and liquid were mixed until reaching a doughy consistency. The Teflon mold was filled with denture-relining material and covered with a polyester film and a glass slide. After polymerization, the denture-relining material was separated from the Teflon mold. Finally, 10 µL of the experimental coating resin was spread on the prepared denture-relining material with a microbrush.A contact angle analyzer  was used in this study to measure the surface wettability of each specimen. Five microliters of distilled water were dropped on the samples randomly, and the contact angles of these specimens were measured after 10 s of contact.The Vickers hardness was investigated using a microhardness tester  under an established load of 0.09 MPa for 20 s using a pyramidal-shaped diamond indenter . MeasureThe color differences were evaluated using a spectrophotometer . A white plate was used for standard calibration before measuring each specimen. The specimens were assessed according to the obtained a*, b*, and L* values, and ΔE* values were calculated according to the CIE Lab system. The color measurements were averaged at various locations on each specimen.Gloss specimens were prepared using a Teflon mold with a 2 mm height and a 10 mm diameter. Two disk-shaped specimens were prepared for each, and eight specimens were used in this study. After fabrication, the specimens were polished sequentially using 800–2000-grit silicon carbide paper. Gloss was analyzed using a Novo-Curve glossmeter . After calibrating the glossmeter, five points on the flat surface of each specimen were randomly measured at an angle of 60°.The samples were sputter-coated with platinum to facilitate the observation of the sample surface. Scanning electron microscopy  images were obtained at an accelerating voltage of 15 kV and a magnification of 50×.Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity, ISO 10993-5:2009. Suspensions of L929 cells were adjusted to a density of 1 × 104 cells/mL, and 100 µL of the cell suspension was incubated with a denture base resin specimen for 24 h [An MTT -2,5-diphenyl tetrazolium bromide) assay was performed using L929 murine fibroblasts  to evaluate the biocompatibility of the control and experimental groups, according to the guidelines established in Candida albicans (ATCC 10231) cultured in a yeast and mold medium  in an incubator at 37 °C for 24 h. The test specimens were sterilized using ethylene oxide. The fungal cultures were diluted to obtain an optical density (OD600) of 0.4–0.6, and the absorbance was measured at 600 nm using an optical density reader . Then, 1 mL of the fungal culture suspension (1 × 108 cells/mL) was placed on each specimen at 37 °C. After 24 h, the samples were washed with distilled water, and the attached fungi were resuspended in 1 mL of YM by sonication  for 5 min. Next, 100 µL of the C. albicans suspension was uniformly spread on YM agar plates, and the number of colony-forming units (CFU) was evaluated.Antifungal tests were carried out using p < 0.05.Five independent replicates were performed for all experiments, and data were calculated as averages and standard deviations using statistical software . The results were analyzed using a one-way analysis of variance followed by Tukey’s statistical test. The significance level was set at According to previous studies, the quality of life related to oral health in the elderly improved after denture prosthesis use, and denture satisfaction was directly related to the quality of life related to oral health . DentureOur previous study reported that the X-ray diffractometry diffraction pattern of Zn-PBG was non-crystalline in nature, consistent with that of a typical glass, and that the distribution was bimodal, as identified with a particle size analysis . The conp > 0.05). It has been reported that when the surface becomes more hydrophobic, the adhesion of bacteria improves [The results of the surface wettability tests are shown in improves . The matimproves .Surface hardness is a key factor that affects the wear resistance of prostheses . When sup > 0.05). The hardness values of the control, 2, 4, and 6 wt% Zn-PBG groups were 27.9 ± 2.1, 29.7 ± 3.2, 30.1 ± 5.2, and 28.4 ± 3.4, respectively.The results of the microhardness tests are shown in Denture color and gloss are also clinically important factors . Color cp > 0.05).The gloss of a material surface is an optical phenomenon expressed as the amount of light reflected from the surface . TherefoAs shown in The first null hypothesis, that a denture-relining material containing zinc-doped phosphate glass would not result in significant differences in the surface properties compared to the control, was accepted.Toxicity due to denture base materials may cause irritation, inflammation, and allergic reactions in oral tissues . It is aAs shown by the MTT assay results in The second null hypothesis, that a denture-relining material containing zinc-doped phosphate glass would not result in significant differences in cell cytotoxicity compared to the control, was accepted.Candida albicans, known to be the pathogen most related to denture stomatitis, was targeted [C. albicans to the surface is an important step in developing denture stomatitis [Fungi readily attach to denture surfaces, and the resulting accumulation of fungi is a major problem for denture wearers . Fungi ctargeted . The adhomatitis ,6,18. Thomatitis . These fomatitis . OverallThe third null hypothesis, that a denture-relining material containing zinc-doped phosphate glass would not result in significant differences in the antifungal activity compared to the control, was rejected.In this study, phosphate-based glass containing zinc was investigated. Zinc possesses antifungal properties, although its mode of action is currently unclear. Previous studies have shown that zinc can induce oxidative stress and cause protein dysfunction in microorganisms. In an earlier study, zinc significantly decreased the number of Gram-negative and Gram-positive bacteria ,24. Its Candida albicans were tested in this study; therefore, there is a limitation in the design of various variables in the experiment. More meaningful results can be obtained if future investigations are conducted using different types of denture base resins and multiple clinically relevant microorganisms. Moreover, a follow-up study to observe the antifungal effect over time is necessary, including the elucidation of a clear antifungal action mechanism for clinical application. The denture base resin developed in this study is bio-friendly and exhibits antifungal activity without changing the properties of the material surface, making it a promising material for denture users in dental clinics.Only one type of denture base resin and Candida albicans. In addition, it was shown that surface properties, such as contact angle, microhardness, color difference, and gloss, were not significantly different compared to the control group. In the biocompatibility tests, the experimental groups did not exhibit cytotoxicity. The present study investigated the antifungal efficacy of a denture-relining material coated with zinc-doped phosphate glass over a short period. Therefore, a long-term investigation is needed. A novel denture-relining material prepared with zinc-doped phosphate glass has potential as an antifungal dental material that is biocompatible while retaining its surface properties without deteriorating.Zn-PBG is a promising antifungal nanomaterial for use as a size-adjustable filler for denture base resins. A denture-relining material coated with zinc-doped phosphate glass showed antifungal efficacy toward


The YFCR genotypes were grown at the International Institute of Tropical Agriculture (IITA) research field, Ibadan. The bioactive constituents , antioxidant , and starch-digesting enzymes (α-amylase and α-glucosidase) inhibitory activities of the products were determined using standard laboratory methods. The glucose response of the products was assessed in human subjects. The concentrations of the bioactive constituents of the products from different genotypes varied significantly (p < 0.05). The ABTS•+ and DPPH• scavenging capacities and the reducing power of the products also differed significantly (p < 0.05), such that the lafun from IITA-TMS-IBA182962, IITA-TMS-IBA070337 and IITA-TMS-IBA070337 had the strongest ABTS•+ and DPPH• scavenging capacities, and reducing power, respectively. The α-amylase and α-glucosidase inhibitory activities of the three products differed significantly (p < 0.05), with the lafun from IITA-TMS-IBA070337 and IITA-TMS-IBA07033 having the strongest α-amylase and α-glucosidase inhibitory activity, respectively. Also, the lafun from IITA-TMS-182986 had the least glucose response, while the fufu from IITA-TMS-IBA070337 had the highest glucose response. Overall, the lafun from different genotypes of YFCR had the most potent antioxidant and starch-digesting enzymes inhibitory activities and the least glucose responses. Hence, lafun may be a promising dietary intervention targeting oxidative stress, hyperglycaemia, and their resultant type 2 diabetes.Yellow-fleshed cassava root (YFCR) is processed into traditional products that may influence its bioactivities. In this study, the antioxidant and anti-hyperglycaemic activities of three traditional products ( The prevalence of complications associated with hyperglycaemia, such as obesity, type 2 diabetes (T2D), cardiovascular diseases, is significantly increasing worldwide. According to IDF , these cConsequently, the association between plant food intake and reduced incidences of complication associated with hyperglycaemia is currently the main focus of some scientific investigations in recent years. This becomes more imperative as the currently available drugs for the treatment and management of some of these complications have been proven to have certain limitations. For example, the clinical use of acarbose for the treatment of T2D is associated with abdominal distention and flatulence in some patients .Provitamin A carotenoids-biofortified (yellow-fleshed) cassava varieties, produced from the biofortification process, serve as a source of energy and important dietary intervention for alleviating vitamin A deficiency, particularly in developing countries . Apart fThus, the purpose of this study was to determine the effect of traditional processing on the antioxidant, starch-digesting enzymes inhibitory activities and glucose response of YFCR genotypes.fufu, lafun, and gari.Samples of five YFCR genotypes , were collected from the International Institute of Tropical Agriculture's (IITA) research farm in Ibadan, Nigeria, from the institute's cassava breeding unit. Each genotype's roots were thoroughly washed with water to remove all dirt and adherent sand particles, and then divided into three 5 kg portions. Each portion was then processed into gari, fufu and lafun is shown in A flow chart for traditional processing of cassava to fufu. YFCR that were freshly harvested were peeled and thoroughly washed with tap water to remove any adhered sand. Approximately 5 kg of peeled YFCR was cut into ~15 cm-long chunks with a stainless-steel hand knife and steeped in 5 liters of water in a plastic bowl for 3–5 days at ambient room temperature (28–32°C) for retting. The retted roots were manually broken and the fibers were sieved out. The mash was manually sieved using a mesh cloth sieve. In a large plastic bowl, sieved mash samples were allowed to sediment for 24 h. Following sedimentation, the water was decanted and the sediment was washed with additional water. The sediment (fufu mash) was dewatered by placing it in Hessian sacks and pressing it with a hydraulic press to remove excess moisture. The pressed mash was then dried for 8 h at 65°C in a cabinet dryer, before being milled in a stainless-steel hammer mill.The traditional processing method described by Sanni et al.  was modiLafun was processed according to Cassbiz  in m/g was calculated from a tannin calibration curve.The concentration of tannin in the products was determined using the method described by Joslyn , with a 2CO3 solution (7.5% w/v) was added. The reaction mixture was shaken and incubated at room temperature for a period of 30 min before the absorbance (at 765 nm) was measured against a blank prepared by dispensing 300 μL of distilled water in place of the sample extract. The total phenolic concentration was presented in mg/g as gallic acid equivalents (GAE).The Folin–Ciocalteu method reported by Chan et al.  was emplThe protocol described by Asha et al.  was used•+ scavenging capacity of the products' extracts was carried out following the method reported by Re et al.  and incubated for 16 h at room temperature in a dark enclosure. After that, the absorbance reading of the ABTS•+ working reagent at 734 nm was adjusted to 0.70 ± 0.02 by diluting with ethanol (95%). A portion of of the ABTS•+ reagent (2.0 mL) and the extract (0.2 mL) was dispensed in a test tube, mixed well and incubated in a dark enclosure for a period of 15 min at room temperature, after which the absorbance reading was taken at 734 nm. ABTS•+ scavenging capacity of each extract, presented in trolox equivalent antioxidant capacity (TEAC), was calculated using a trolox calibration curve.The ABTSe et al. . To prep• scavenging capacity of the products' extract was conducted by adopting the procedure described by Cervato et al.  was incubated in a dark enclosure at room temperature for a period of 30 min. Thereafter, the absorbance reading at 517 nm was taken and the DPPH• scavenging capacity of the extract, presented as SC50 (extract concentration that scavenged 50% of DPPH•).The DPPHo et al. . Vitamin3 solution. The sample extract (2.5 mL) was mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6), and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for a period of 20 min, following which 2.5 mL of 10% trichloroacetic acid was added. The mixture was spinned at 650 × g for a period of 10 min, and the supernatant was divided into 2.5 mL portions in different test tubes. Next, distilled water (2.5 mL) and 0.1% ferric chloride solution (1 mL) were added to the content of each test tube, and the absorbance reading at 700 nm was taken. The reducing power of the extracts, presented as gallic acid equivalent in mg per gram, was calculated using a gallic acid calibration curve.The protocol reported by Oyaizu  was adopThe inhibition of alpha-amylase by the products' methanolic extract was determined using the method described by Kwon et al. . In thisWhere AR is the mean absorbance reading of the reference; AS is the mean absorbance reading of the sample.2CO3 (2 mL). The absorbance of the yellow p-nitrophenol released from the hydrolysis of PNPG was read at 400 nm. The percentage α-glucosidase inhibition was calculated using the following equation:The protocol described by Kim et al.  was adopWhere AR is the mean absorbance reading of the reference; AS is the mean absorbance reading of the sample.Ethical clearance (MOH/KS/EU/777/497) to conduct the glucose response experiment was obtained from Kwara State Ministry of Health, Ilorin, Nigeria. Nineteen apparently healthy, non-diabetic and non-obese subjects, comprising 8 male and 11 female students of Kwara State University, Malete, Ilorin, Nigeria, aged 18–25 years, volunteered to participate in the study. Informed consent was obtained from the volunteers.fufu, lafun and gari). Next, 50 g of reconstituted fufu/lafun/gari was served (on different days) with an equal portion of ewedu soup and fish. The formula and mode of cooking the soup were consistent throughout the study.All the volunteers were first subjected to an oral glucose challenge after overnight fasting by drinking 50 g of glucose in 25 mL of potable water. Their blood glucose level was measured at 0, 30, 60, and 90 min using a glucometer (Accu-Chek Active). After that, they were randomly distributed into groups of three volunteers per sample . In addition, GraphPad Prism 5 for Windows, GraphPad Software, San Diego, California USA, fufu, lafun and gari processed from the five YFCR genotypes  are presented in gari from IITA-TMS-IBA070337 and fufu from IITA-TMS-IBA182986, respectively. Further, the total carotenoid content of fufu from IITA-TMS-IBA182986 was significantly (p < 0.05) higher than those of fufu, lafun and gari from the other four genotypes . The total phenolics level ranged from 0.1 to 0.38 GAE mg/g in lafun from IITA-TMS-IBA182986 and gari from IITA-TMS-IBA182962, respectively. Tannins content was in the range of 0.003–0.019 TAE mg/g in fufu from IITA-TMS-IBA070337 and lafun from IITA-TMS-IBA182962, while the total flavonoids ranged from 0.03 to 0.24 QE mg/g in fufu and lafun from IITA-TMS-IBA070337 and gari from IITA-TMS-IBA182961, respectively. Generally, the levels of the bioactive constituents of the products varied significantly (p < 0.05) across the genotypes.The bioactive constituents  of the in vitro antioxidant activity of fufu, lafun and gari processed from five YFCR genotypes are presented in •+ scavenging ability ranged from 5.96 to 8.82 TEAC mmol/g in fufu from IITA-TMS-IBA182961 and lafun from IITA-TMS-IBA182962, respectively. There was a significant difference (p < 0.05) in the ABTS•+ scavenging ability of the three products across the genotypes. The reducing power also varied significantly (p < 0.05) among the products from the different genotypes and ranged from 0.34 to 0.97 GAE mg/g in fufu from IITA-TMS-IBA070337 and lafun from IITA-TMS-IBA182962, respectively. The DPPH• scavenging ability, expressed as the products' extract concentration that scavenged DPPH• by 50% (SC50) also varied significantly (p < 0.05) among products from different genotypes and ranged from 85.61 to 398.30 mg/mL in lafun from IITA-TMS-IBA182961 and fufu from IITA-TMS-IBA07033, respectively.The fufu, lafun and gari from the five genotypes of YFCR are shown in 50 differed significantly (p < 0.05) among the three products from the different genotypes and ranged from 24.57 to 83.88 mg/mL in lafun from IITA-TMS-IBA070337 to lafun from IITA-TMS-IBA182962, respectively. On the other hand, the α-glucosidase IC50 ranged from 32.36 to 62.56 mg/mL in lafun from IITA-TMS-IBA07033 and lafun from IITA-TMS-IBA182961, respectively.The α-amylase and α-glucosidase inhibitory activity of extracts of the fufu, lafun and gari from the YFCR genotypes are shown in lafun from different genotypes had the lowest glucose response, increasing the blood glucose by +5 mg/dl at the end of 19 min.The blood glucose responses resulting from the consumption of the fufu, lafun and gari from the five genotypes of YFCR in this study varied among the products. This agrees with the report of Maziya-Dixon et al.  previously reported for uncooked fufu produced from three genotypes  of yellow-fleshed cassava . Maziya-Dixon et al.  of the fufu, lafun and gari suggests that they may help alleviate the Fe2+-mediated generation of hydroxyl radical (OH•) from hydrogen peroxide (H2O2)  , and Fe2xidation , therebylafun had the strongest antioxidant activity, while fufu had the weakest antioxidant activity. The variations in the antioxidant activity of the products from different the YFCR genotypes might be attributed to their different processing methods  (Fe3+) to iron (II) (Fe2+) might be attributed to bioactive constituents, including carotenoids and polyphenols, which are known to possess antioxidant activity  is a well-established strategy for determining the ability of food products to lower postprandial blood glucose response. It is indicative of the anti-hyperglycaemic activity of the food . Alpha-aafun having the strongest inhibitory effect on both α-amylase and α-glucosidase. As explained for the antioxidant activity of the products, this variation may be due to the impact of their different processing methods. Reducing the hydrolysis rate of starch through the inhibition of digestive enzymes (α-amylase and α-glucosidase) is an important strategy for relieving postprandial hyperglycaemia  lower compared to that of the standard glucose. The variation in the glucose response of these different products also agrees with a previous report that different processing methods increased the digestibility of starch in cell walls and plant polysaccharides and conversely reduced their resistance to enzymatic action during digestion  displayed appreciable antioxidant and starch-digesting enzymes inhibitory activities in vitro. Overall, the lafun from different genotypes had the strongest antioxidant, α-amylase and α-glucosidase inhibitory activities. The lafun from IITA-TMS-182986 also had the least glucose response, while fufu from IITA-TMS-IBA070337 had the highest glucose response. Therefore, lafun may be a preferred traditional product of YFCR genotypes for dietary intervention, targeting oxidative stress and postprandial hyperglycaemia. Further study is recommended to ascertain the mechanisms and interactions of the various bioactive constituents in lafun in mediating the antioxidant and anti-hyperglcaemic activities observed in this study.The The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Kwara State Ministry of Health, Ilorin, Nigeria with Ethical clearance number MOH/KS/EU/777/497. The patients/participants provided their written informed consent to participate in this study.EAl and EI: conceptualization. EI, EAl, BK, and MA: methodology. EI and BK: software and writing—original draft preparation. BK and MA: formal analysis, investigation, and data curation. AA, BM-D, EP, EAl, and EAj: resources and writing—review and editing. EI, EAl, and EAj: supervision. EAl and AA: project administration. EAl, AA, EP, and BM-D: fund acquisition. All authors have read and agreed to the published version of the manuscript.This research and the APC were funded by the CGIAR Research Program on Roots, Tubers and Bananas (RTB) and the Bill & Melinda Gates Foundation (BMGF) through OPP1019962.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The current COVID-19 pandemic has highlighted the necessity of more efficient antiviral compounds. The antiviral efficacy of adenosine-based analogs, the main repurposed drugs for SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition, is mainly assessed through in vitro or cell-free polymerization assays, under arbitrary conditions that do not reflect the physiological environment. We show that SARS-CoV-2 RdRp inhibition efficiency of remdesivir and cordycepin, two common adenosine analogs, is influenced by endogenous adenosine level, and that the current clinically approved concentrations for COVID-19 treatment are suboptimal for effective RdRp inhibition. Furthermore, we identified GTP as the rate-limiting nucleotide of SARS-CoV-2 replication. Our results demonstrate that nucleotide sensitivity of the RdRp complex and competition of nucleoside analog drugs against endogenous concentrations of nucleotides are crucial elements to be considered when designing new SARS-CoV-2 antiviral compounds. Recent severe infectious disease outbreaks caused by coronaviruses, such as the 2003 severe acute respiratory syndrome outbreak caused by SARS-CoV-1, the 2012 Middle Eastern respiratory syndrome outbreak caused by MERS-CoV .50 values by underestimating the intracellular levels of the active triphosphate (TP) form. Remdesivir (GS5734) is a prodrug of a 1′-cyano-substitued adenosine analog, and it was shown to inhibit SARS-CoV-2 in Vero E6 cells with IC50 ranging from 0.77 μM [50 of 0.77 μM [50 of up to ~450 μM based on the effective intracellular concentration of the active compound GS-443902. Such high intracellular concentrations should be capable of a complete inhibition of replication, but the clinical effectiveness was rather sobering [Targeting the RdRp is an effective strategy to inhibit viral replication ,6. Remde 0.77 μM  to 26.9  0.77 μM . These v 0.77 μM . Considesobering . A possisobering  are ofteThe key obstacle of NAs in efficiently inhibiting the viral polymerization reaction is the competition with the endogenous pool of nucleosides. This can be overcome by inhibition of de novo synthesis of pyrimidines or purines , which iHere, we use radioactive elongation assays to demonstrate how adenosine-based NAs are competing against a varying concentration of NTP pool added to the reaction. Additionally, we report GTP as the rate-limiting nucleotide of SARS-CoV-2 replication. These findings explain why some NAs are not as efficient as expected and give a direction for future NA design that should be based on guanosine.32P-ATP labeled primer , initially found to inhibit the RdRp of the Ebola virus , can be Here, we investigated two characteristics leading to decreased NA efficiency. First, we showed that NAs have to compete against the NTP pool, and that endogenous NTPs have a higher incorporation kinetics compared to the NAs that we have tested. Second, we demonstrate that GTP is the rate-limiting nucleotide for the SARS-CoV-2 replication complex.50 of 6.56 μM in their in vitro study, similar to our determination of 7.81 μM in the presence of 1 μM NTPs  is lost with increased concentration of the NTP pool. This effect is also true if we deplete the direct competitor ATP and force the incorporation of the NA. Thus, the remaining NTPs are able to compete against the NA after reaching a certain concentration.6 higher than that of their infected hosts [Most viral RNA and DNA polymerases are known to catalyze non-Watson–Crick base pairing ,25,26,27ed hosts ,29,30. Sed hosts . The Exoed hosts ,32.32P-GTP [To investigate the fidelity of the pure RdRp complex, we systematically depleted each single NTP. This led to the identification of GTP as the rate-limiting nucleotide of the SARS-CoV-2 RdRp complex. This was possible because our assay design was based on a radiolabeled oligonucleotide as a primer, while initial studies were mainly based on the incorporation of radioactive NTPs such as 32P-GTP . This asTo conclude, our study demonstrates for the first time (to our knowledge) the inhibitory effect of cordycepin-TP on the RdRp of SARS-CoV-2 in an in vitro polymerase assay. Therefore, the ProTide NUC-7738 could be considered as a drug for further repurposing studies in in vivo settings and as starting point for further structural developments.We propose that in designing drugs against RdRps, researchers should include the discovered biochemical properties presented here, namely that NAs should be based on guanosine, and that the intracellular concentrations due to active uptake and concentration of endogenous competitors, together with the ultrastructural features, could streamline the effort to find potent drugs that are effective in the lowest concentrations to avoid any adverse effects. Further, one should consider combinatory studies of these newly derived NAs with inhibitors of de novo nucleotide synthesis to explore synergistic effects. The dynamics of viral mutations and newly arising strains emphasizes the need for efficient drugs to bridge the time gap for vaccine adaptations.The expression constructs for nsp7, nsp8 and nsp12 and protein purification protocols are as described previously .32P-ATP . Subsequently the labeled oligonucleotide was purified by denaturing urea acrylamide gel electrophoresis. The purified primer was annealed to the 40 nt RNA template (5′-CUAUCCCCAUGUGAUUUUAAUAGCUUCUUAGGAGAAUGAC-3′) (IDTDNA) in 10 mM Tris-HCl, pH 7.5, 100 mM KCl. A typical 10 µL reaction was performed in elongation assay buffer 20 mM Tris-HCL, pH 8, 5 mM MgCl2, 5 mM DTT, 5% glycerol, with 500 nM total protein concentration  and 250 nM annealed RNA. The inhibitors GS-443902  and cordycepin-TP  were added and shortly incubated before the reaction was started by the addition of NTPs in concentrations as indicated in the figures. The reaction was stopped by the addition of 2×PAA-loading buffer and analyzed by denaturing urea acrylamide gel electrophoresis and phosphorimaging.The polymerization assay was conducted as described previously , with mi50 was determined by transforming the recorded radiographs to intensities using the ImageJ software . These values were used to derive the IC50 by a sigmoidal dose–response curve using the GraphPad Prism software .The IC


Collagen and chitosan are frequently used natural biomaterials in tissue engineering. However, most collagen is derived from animal tissue, with inconsistent quality and pathogen transmittance risks. In this context, we aimed to use a reliable Type-III recombinant human collagen (RHC) as an alternative biomaterial together with chitosan to develop novel photo-responsive bioinks for three-dimensional (3D) bioprinting. RHC was modified with methacrylic anhydride to obtain the RHC methacryloyl (RHCMA) and mixed with acidified chitosan (CS) to form composites CS-RHCMA. The characterizations demonstrated that the mechanical properties and the degradation of the bioinks were tunable by introducing the CS. The printabilities improved by adding CS to RHCMA, and various structures were constructed via extrusion-based 3D printing successfully. Moreover, in vitro tests confirmed that these CS-RHCMA bioinks were biocompatible as human umbilical vein endothelial cells (HUVECs) were sustained within the constructs post-printing. The results from the current study illustrated a well-established bioinks system with the potential to construct different tissues through 3D bioprinting. The latest progress includes the development of bioinks from advanced biomaterials and their use in 3D bioprinting. Designing advanced bioinks always relies on the synthesis of polymers or modification of natural polymers. Collagen , gelatinCollagen  and chitThis study adopted a reliable, predictable, and chemically-defined Type-III recombinant collagen (RHC) developed in the Bioengineering Lab of Nanjing University of Science and Technology together with commercial chitosan (CS) to fabricate 3D printable bioinks. We aimed to explore the feasibility of using these CS-RHCMA as reliable bioinks in tissue engineering. These CS-RHCMA bioinks and their UV-cured constructs were then comprehensively investigated. Fourier transform infrared spectroscopy (FT-IR) was used to determine the success of producing the RHCMA through MAA modification. The internal morphology of the UV-cured bioinks was investigated using scanning electron microscopy (SEM) after freeze-drying. The mechanical strength, as well as the biodegradation of the UV-cured bioinks, was carefully determined. Lattices were printed by extrusion-based 3D printing to evaluate the printability of the bioinks, and the viscosity values of the bioinks were assessed. The cytotoxicity of the bioinks was assessed by the sample elution test according to ISO-10993:5. HUVECs were pre-loaded in CS-RHCMA bioinks and printed to form cell-laden constructs where cell survivability and morphology were determined. Our results indicated that the bioinks based on chitosan and RHC demonstrated tunable mechanical properties. These bioinks showed ideal printability compared to RHCMA only bioinks and had no toxicity to the cells. The 3D printed constructs also showed their capability to maintain the viability of HUVECs, thus laying the foundation for its application in the tissue engineering field in the future.Type III recombinant human collagen  with high hydrophilicity was prepared by our laboratory . MethacrA schematic diagram of the bioinks preparation is shown in w/v) RHC phosphate solution, stirred for 1 h, and then centrifuged to collect the supernatant. Deionized water was added to dilute the supernatant, and dialysis was conducted for 4 d at 37 °C with a dialysis bag  to filter the unreacted methacrylic anhydride. The pH value was adjusted to 7, and the solution was freeze-dried to collect the MAA-modified recombinant human collagen (RHCMA) powder.To obtain the RHC methacryloyl, 4.8 mL MAA was added dropwise into 40 mL 15% (w/v) CS and 10% (w/v) RHCMA solution, respectively. The CS and the RHCMA solution were mixed at mass ratios of 1:3, 2:3, and 3:3, and final concentrations were set to 10% (w/v). The pH of the mixtures was carefully adjusted to 7 with 1 M sodium hydroxide. An amount of 0.25% (w/v) LAP was added to these mixtures; they were stirred in the dark for 1 h at room temperature, thus preparing three types of photo-responsive CS-RHCMA bioinks.The CS and RHCMA powder were dissolved in PBS solution to prepare the 1% . All spectra were recorded in the absorption mode at 2 cmThe scanning electron microscope  was adopted to observe the internal morphology of UV-cured CS-RHCMA bioinks. Before the SEM observation, the freeze-dried samples were sectioned using a scalpel and sputter-coated with gold for 90 s. The average pore size was calculated by measuring the diameters of randomly selected pores.The mechanical properties of the UV-cured bioinks were evaluated via the stress-strain test with a universal mechanical test system . The cylindrical samples with a diameter of 20 mm and a height of 5 mm were compressed at a constant speed of 10 mm/min up to 70% strain. The test instrument collected the force and deformation data and then converted them into stress-strain values. The compressive modulus was determined according to the slope of the linear portion of the 10% strain for all samples.For biodegradation tests, UV-cured bioinks samples were subjected to either PBS buffer (pH 7.2) or PBS buffer containing lysozyme . Samples were weighed and then immersed in the test solution to determine their degradation behavior. The solution was exchanged every 2 days, and the incubation temperature was set to 37 °C. At each defined time point, samples were removed from the solution and rinsed with distilled water. Filter paper was used to remove excess water, and samples were weighed immediately. The weight changes that represented the biodegradation status were calculated as follows: (Wt − Wo)/Wo × 100%, where Wo is the initial weight of the UV-cured samples and Wt is the weight of the degraded samples at a specific time point .2) light source was adopted to solidify the printed constructs. Printability is defined based on a square shape using the following equation quoted from a previous study: Printability = L2/16A, where L is the perimeter of the single grid, and A refers to the area within this grid [The viscosity values associated with the rheological properties of the bioinks were determined by a rheometer  equipped with a 25 mm plate (CP-25), and tested with an angular frequency of 10 rad/s. The gap distance was 50 μm; all tests were conducted at room temperature. To determine the printability of the CS-RHCMA bioinks, grid structures were printed by an extrusion-based 3D bioprinter  equipped with a 25G nozzle. The printing speed was 16 mm/s, and the extrusion volume was 0.07 μL/mm. A 405 nm UV  and antibiotics (100 IU/mL penicillin and 100 ug/mL streptomycin) was used as the culture medium for the HUVECs, and cells were cultured at 37 °C in a 5% CO2, and cultured at 37 °C in a 5% CO2 humidified incubator. After one day of culture, the medium was replaced with extracted eluents. Cells cultured in the fresh medium were used as a control. An amount of 20 uL CCK-8 was added to each sample at defined culture times (24 h and 72 h) and incubated for another 3 h. A microplate reader  was used to determine the absorbance at 450 nm. According to ISO 10993:5, the sample was considered cytotoxic if the cellular viability was reduced to less than 70% of the control group. The status of the HUVECs was examined by AO/EB double-fluorescent staining. The AO stained viable cells and exhibited a bright green intact structure, whereas the EB stained apoptotic cells and appeared as a red-orange color. Cellular morphology was observed under an inverted fluorescence microscope .The cytotoxicity of the CS-RHCMA was evaluated via a modified elution test according to the ISO-10993:5. First, 0.5 mL bioinks were added into each well of a 12-well plate and then cured using UV irradiation. Next, each sample was taken out and immersed in 2.5 mL F-12K. The elution was collected after incubating for 24 h at 37 °C. Meanwhile, HUVECs were seeded in the 96-well plate with a density of 3000 cells/cm6 cells/mL. Grid constructs with 500 µm thickness were then printed with the same parameters in the printability tests and UV-cured for 45 s. The solidified constructs were incubated in a fresh medium. AO/EB double-fluorescent staining was used to identify the viability of the HUVECs within the constructs at 1 h and 48 h post-printing . The survivability of the HUVECs post-printing was determined by ImagePro Plus from the captured fluorescent images. The ImagePro plus software was used to identify the green living cells and red dead cells.HUVECs were collected firstly and mixed with CS-RHCMA to obtain bioinks with a cell density of 1 × 10p < 0.05 and ** p < 0.01, where p > 0.05 was considered not statistically significant.At least three samples of each group were tested. Data collected from each experiment are presented as mean ± standard deviation and plotted with OriginPro 2017 . Differences were detected by the t-test; significance is indicated with * −1 region corresponds to the O-H and N-H stretching. The band confirmed the presence of a residual at around 1650 cm−1 (C=O stretching of Amide I) which represents the residual N-acetyl groups, and the absorption band at 1150 cm−1 attributed to the asymmetric stretching of the C-O-C bridge [2 protonation influenced the hydrogen bond among chitosan molecules. For the RHC molecule, a typical collagen band was found at 1625 cm−1, corresponding to the C=O stretching vibrations of Amide I. Amide II was found at 1545 cm−1, corresponding to N-H deformation vibrations. Amide A was found at 3290 cm−1, corresponding to N-H and O-H vibrations. υC-H corresponding to amide B was found at 3077 cm−1. The band at 1046 cm−1 was attributed to the C=C-H in-plane bending vibrations of RHCMA, indicating that methacryloyl substitutions occurred on the RHC molecules. Finally, the intensity of asymmetric stretching varied with CS to RHCMA ratios  by introducing the acidified CS to the RHCMA bioinks, it confirmed our CS-RHCMA bioinks’ desired printability. Viscosity is one of the key parameters to the printability in extrusion-based bioprinting [To understand the rheological property of the bioinks, the viscosity values of the bioinks were determined. printing , and higprinting . Our resprinting . Construp > 0.5) compared to the control group at the selected time points (To determine the cytotoxicity of the CS-RHCMA bioinks, cell morphology and survivability were evaluated by AO/EB double-fluorescent staining. As shown in e points B. FurtheThe CS-RHCMA bioinks were adopted to load HUVECs for bioprinting, then the printed constructs were UV-cured for 45 s immediately. The HUVECs within the printed structures were visualized by AO/EB double-fluorescent staining, as shown in This research added the acidified chitosan to RHCMA solution to prepare photo-responsive bioinks for 3D printing. The mechanical strengths and internal pore size of the CS-RHCMA bioinks formed constructs that were tunable by varying the CS to RHCMA ratio. The biodegradation ability of these bioinks formed constructs was also influenced. Introducing CS to RHCMA improved the printability of the bioinks as 3D constructs were well-built through extrusion-based 3D printing. More importantly, these CS-RHCMA bioinks demonstrated great biocompatibility that supports the viability of the HUVECs within the printed constructs. In conclusion, the developed CS-RHCMA bioinks in the current study showed their potential in 3D bioprinting and tissue engineering. It is hoped that a complicated tissue structure with biological activities will be constructed in the future.